Dissecting The Molecular Recognition Of Innate Immune Toll-like Receptor 4 And Toll-like Receptor 13 Modulators

Toll-like receptors,as a kind of innate immune receptors,are in close relationship with the occurrence and progression of many diseases.As one of the most important Toll-like receptors,Toll-like receptor 4(TLR4)can recognize pathogen-associated molecular patterns in bacteria and viral,and is the pivotal target for the treatment of many diseases,including sepsis,neurodegeneration diseases,cancer,etc.However,there are limitations in the clinical application of current TLR4 modulators.On the one hand,there is a lack of potent TLR4 small-molecule antagonists in the treatment of diseases like sepsis.On the other hand,small-molecule agonists of TLR4 were spotted with species-specific effects and failed to activate human TLR4,making them unable to be applied clinically as adjuvants.Therefore,the exploration of effective TLR4 modulators is in great need.Toll-like receptor 13(TLR13)was discovered to recognize the PAMPs of bacteria until recent years,yet few studies focused on its recognition and interaction mechanism with ligands.It has been reported that a specific sequence of 23S rRNA,RNA2054-2068(ACG GAA AGA CCC CGU),can bind to and activate TLR13,leading to the activation of downstream signaling pathways and the release of inflammatory factors,such as interleukin-1β.However,it is still unclear the existing form of RNA and how TLR13 recognizes RNA15 at the atomic level.Therefore,this dissertation takes TLR4 and TLR13 as the research object,investigating the recognition and interacting mechanism between Toll-like receptors and their small-molecule and biomacromolecule modulators.The main results are as follows:1.Through repositioning existing FDA-approved drugs,pentamidine,an antiprotozoal drug,was identified as an antagonist of TLR4.The mechanism and function of pentamidine were investigated gradually in the dimension of atomic,molecular,cellular,and individual levels.The results indicated that pentamidine can bind to the myeloid protein 2(MD2),the adaptor protein of TLR4,and decrease the thermal stability of MD2.What’s more,pentamidine was able to inhibit the increased release of inflammatory factors induced by LPS,and the activities of derivatives of pentamidine in inhibiting TLR4 were correlated with their hydrophobicities.Further studies indicated that pentamidine suppressed the activation of NF-κB and MAPK signaling pathways downstream of TLR4 induced by LPS.Animal models of sepsis indicated that pentamidine alleviated organ inflammation and decreased the mortality of septic mice.Therefore,this study not only proved that pentamidine has the potential to be a candidate drug in clinical use for the treatment of sepsis but also provide a direction for the subsequent optimization and modification of pentamidine in improving its TLR4 inhibiting activities.2.As a novel small-molecule agonist for mouse TLR4/MD2,Neoseptin 3 fails to activate human TLR4/MD2,while the underlying mechanism is unclear.Herein,molecular dynamics simulations were performed to investigate the species-specific molecular recognition of Neoseptin 3.Lipid A,a classic TLR4 agonist showing no apparent species-specific sensing by TLR4/MD2,was also investigated for comparison.Neoseptin 3 and lipid A showed similar binding patterns with mouse TLR4/MD2.Although the binding free energies of Neoseptin 3 interacting with TLR4/MD2 from mouse and human species were similar,protein-ligand interactions and the details of the dimerization interface were substantially different between Neoseptin 3-bound mouse and human heterotetramers at the atomic level.Neoseptin 3 binding made human TLR4/MD2 more flexible than lipid A-bound human TLR4/MD2,especially at the TLR4 C-terminus and MD2,which drives human TLR4/MD2 fluctuating away from the active conformation.In contrast to Neoseptin 3-bound mouse TLR4/MD2 and lipid A-bound mouse/human TLR4/MD2 systems,Neoseptin 3 binding to human TLR4/MD2 led to the separating trend of the C-terminus of TLR4.Furthermore,the protein-protein interactions at the dimerization interface between TLR4 and the neighboring MD2 in the Neoseptin 3-bound human TLR4/MD2 system were much weaker than those of the lipid A-bound human TLR4/MD2 heterotetramer.These results explained the inability of Neoseptin 3 to activate human TLR4 signaling and accounted for the species-specific activation of TLR4/MD2,which provides insight into transforming Neoseptin 3 as a human TLR4 agonist.3.As a biomacromolecule agonist for TLR13,RNA2054-2068(ACG GAA AGA CCC CGU)binds TLR13 in a stem-loop-like manner,yet its recognition mechanism was unclear.A series of experiments in the measurement of fluorescence and anisotropy indicated that RNA15 existed mainly in the form of hairpin in solutions,and the hairpin form increased its stability against the digestion of nuclease.Energy conversions of different recognition processes were calculated via conventional molecular dynamics simulations and umbrella samplings subsequently.Through the energy analyses,the most favored recognition mechanism of RNA15 by TLR13 was found.Few stem-loop-like RNA,which existed in balance with hairpin RNA15,bound to the preassembled inactive TLR13 dimer,thus leading to the activation of TLR13.This study calculated the energies of different recognizing pathways between RNA15 and TLR13,providing a paradigm for investigating the dynamic recognition mechanism of TLRs by their ligands via molecular dynamics simulations.In summary,this dissertation takes Toll-like receptor 4 and Toll-like receptor 13 as research targets,utilizing biological methods,chemical methods,and computational simulation methods to dissect the recognition process and interacting mechanisms of Toll-like receptors with their small-molecule and biomacromolecule modulators in atomic,molecular,cellular,and individual levels,respectively.And it will not only provide guidance and direction for further modifications and optimizations of smallmolecule TLR4 modulators,supplying candidate drugs for the treatment of related diseases but also served as a paradigm for investigating the recognition and interaction mechanism of ligands to Toll-like receptors via molecular dynamics simulations.